Infant formulas are commercially available in a variety of forms including ready-to-feed, concentrated liquid and powdered forms. Infant formulas typically contain casein and/or whey proteins intended to ensure that the infant fed the formula receives adequate amounts of amino acids and, in particular, the essential amino acids for proper nutrition. Two factors in determining the nutritional quality of food proteins are digestibility and bioavailability. Typically, these formulas contain a higher level of protein than the level found in human breast milk. Infant formulas are manufactured with higher levels of proteins to account for the assumed lower digestibility of the proteins.
Studies of infant formulas have shown that the processes used during the manufacture of these formulas have nutritional consequences such as lowered solubility and/or digestibility of the proteins in the formula. For example, heat treatment over extended periods of time that is used to produce concentrated liquid and ready-to-feed infant formulas has been shown to decrease digestibility of proteins. As a result of exposure to heat, proteins denature or aggregate, possibly altering their digestibility. The treatment of milk at high temperatures has also been studied and has been shown to increase reactions of amino acids with sugars known as Maillard reactions. These reactions have been shown to decrease the bioavailability of amino acids by limiting the accessibility of proteolytic enzymes.
In vivo protein digestion is a two-step process. The first step is exposure of the protein to the pre-digestive enzyme pepsin. The second step involves hydrolysis with pancreatic enzymes. The evaluation of amino acid availability in vivo is difficult because protein digestion products are carried quickly into, and absorbed by, the small intestine. Additionally, endogenous proteins may be present and may be digested and absorbed at rates different from proteins ingested as food or in the form of a dietary supplement. Therefore, in vitro analyses of the digestibility of proteins have been developed.
The evaluation of infant formula digestibility has been performed by enzymatic hydrolysis colorimetric analysis (degree of hydrolysis using TNBS) and size exclusion chromatographic techniques such as high performance liquid chromatography (HPLC). The accuracy and precision of the information provided by these approaches is compromised by the presence of insoluble protein and/or by spectrophotometric and chromatographic interferences.
In vitro digestions of infant formula have been conducted using the pre-digestive enzyme, pepsin, and pancreatin. The digestibility of proteins was determined by measuring the increased level of non-protein nitrogen (NPN) following the in vitro digestion process as determined by Kjeldahl analysis. However, the enzymes used in these studies included no standardization of enzyme activity and therefore the activity of the enzymes used in the digestion may vary significantly from lot to lot. Further, nitrogen analysis by Kjeldahl procedures lack specificity in the quantification of digested and undigested protein. Specificity in the types of amino acids digested provides better guidance as to the required formulations of nutritional products, including infant formula, to further ensure digestion and absorption of essential amino acids by infants who are fed the formula.
Digestive studies have included assays to determine the activity levels of proteins such as pepsin, trypsin and chymotrypsin used in the digestion process. Pepsin activity has been measured in terms of units of trichloroacetic acid (TCA)-soluble products. Trypsin and chymotrypsin activities have been measured in terms of hydrolysis rate for a particular amino acid. While determining the activity level of the enzymes to be used could improve standardization of such assays, the added steps of determining enzyme activity are cumbersome and time consuming.
There is a need for a method of in vitro protein digestibility determination that utilizes enzymes having standardized activity. There is further a need for the results of such method to provide specificity in amino acid digestibility.
The invention provides a method for determining the digestibility of proteins while providing specificity in the quantification of digested and total (digested and undigested) protein.